From owner-cellbiol@net.bio.net Mon Jan 27 22:00:00 1997 Path: biosci!daresbury!is.bbsrc.ac.uk!news From: Colin Farquharson Newsgroups: bionet.cellbiol Subject: Percolls-cell clumping Date: Tue, 28 Jan 1997 15:49:26 +0000 Organization: BBSRC Biotechnology and Biological Sciences Research Council Lines: 16 Message-ID: <32EE2006.4C07@bbsrc.ac.uk> Reply-To: Roslin, Institute, Roslin, EH25, 9PS NNTP-Posting-Host: pc0757.ri.bbsrc.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) I am trying to separate chondrocytes into their different sizes using Percoll (Pharmacia) discontinuous gradients (1.04 - 1.08). 2mls of ech are gently layered on top of each other and the cells suspended (20-40 million) in 1 ml of medium on top. Spun in a swing out motor at 400g for 30 min at RT. Things often works but often there is extensive clumping at the 1.06 band and this results in poor separation. Cells appear to be 'dragged down' from their band above and cells that should move further down are inhibited from doing so. Has anyone else had trouble with cells clumping together and anyone know how to resolve it ? Colin Farquharson Roslin Institute Edinburgh Scotland .